Both HIV-1 and METH can cause behavioral changes and brain injury, but their interaction is poorly understood. In the current funding period, we observed that a one-time METH binge caused a reduction in hippocampal post-tetanic potentiation only in HlV/gp120-transgenic (tg) but not control mice. RNA expression studies indicated that both HIVgp120 and METH trigger significant changes of glutamatergic and GABAergic neurotransmission. In vitro studies showed that METH at 100 pM increased neurotoxicity of HIVgp120 and excitotoxic NMDA. METH also increased HIV infection of human macrophages in association with down-regulation of at least four interferon-inducible genes. Therefore, the overall hypothesis is that use of METH aggravates behavioral disturbances and neurotoxicity associated with HIV-1 infection. Our Specific Aims are to: (1) identify affected neural networks by studying gene expression associated with long-term effects of METH in HlVgp120- and iTat-tg and non-tg control mice and to relate such gene expression changes to behavioral performance; (2) determine whether METH interferes with the efficacy of cART drugs to reduce viral replication and production of neurotoxic products by infected monocytes/macrophages. For Aim 1, gp120- and iTat-tg and non-tg control mice will be treated with a novel chronic, low dose 12-week METH regimen. Following a 5-month abstinence period and behavioral testing, neuronal and glial injury and gene expression will be analyzed in brain tissue using deconvolution microscopy, microarray and qRT-PCR. Gene expression data will be analyzed separately for cortex, hippocampus, striatum and other brain regions, and correlated with behavioral outcomes and neuropathology. Follow-up studies to gene expression analyses will probe protein expression, localization and function using biochemical and microscopy approaches. For Specific Aim.2, macrophages infected with HIV will be treated with ARV combinations and varying METH concentrations. The neurotoxicity of supernatents from such macrophage experiments will be tested on rat microglia depleted mixed neuronal glial cerebrocortical cells. The long-term objective is to identify neurotoxic mechanisms that are potential targets for therapeutic intervention.